Carotenoid and carotenoid esters: analysis and in vitro digestion in individual and co-consumed foods

The interest in understanding the fate of carotenoid and carotenoid esters through the digestion process and its impact in human health is growing, making their bioaccessibility assays one of the most innovative issues of study in the field of Food and Nutrition. Challenging aspects related to the analysis of carotenoids and carotenoid esters in foods, which is a requirement for assessing their bioaccessibility, and to the in vitro digestion methods used for this purpose were addressed in this thesis. The identification of the native carotenoid profile of foods is often overlooked because of the complexity of such analysis. Among other factors, interfering lipids that remain in the extract when no saponification step is carried out impair or even preclude the compound identification. In this sense, a new pre-chromatographic two-step cleanup procedure was developed to make feasible the identification of the native carotenoid composition of murici, an Amazonian fruit. Interfering compounds (mainly triacylglycerides) were efficiently removed after physical separation followed by open column chromatography, thereby allowing the identification of 35 carotenoids (six free carotenoids, 14 monoesters and 15 diesters) in non-saponified extracts from murici by HPLC-DAD(APCI)MS/MS, whereas only 6 compounds were identified when no cleanup procedure was performed. In addition, a recent publication of a new in vitro digestion method as an international consensus by the INFOGEST action drew attention to the necessity for standardization of the in vitro digestion conditions of foods at the international level. The INFOGEST method, however, does not address crucial steps needed to assess the carotenoid bioaccessibility, and it is more laborious, time-consuming and expensive than the traditionally used ones. Therefore, the INFOGEST method was adapted for carotenoid analysis by coupling the steps of micellar fraction separation and carotenoid extraction from the micelles, which allowed the successful determination of the in vitro bioaccessibility of carotenoid esters in murici and bioaccessibility of carotenoids in a large group of carotenoid-rich foods. The results obtained with the adapted INFOGEST method were compared to those found using relatively simple and consolidated in vitro digestion models, traditionally used to estimate the bioaccessibility of carotenoids and carotenoid esters. The adapted INFOGEST method provided values of carotenoid and carotenoid ester bioaccessibility in murici higher than the results obtained using the digestion method that was being used in our laboratory, published in 2014. The carotenoid bioaccessibility ranged from 4 to 29%, depending on the digestion method and carotenoid structure, and free carotenoids overall presented higher efficiencies of micellarization than free carotenoids and monoesters. Moreover, compared with the first in vitro digestion method adapted for carotenoids, published in 1999, the adapted INFOGEST method generally provided similar estimates of carotenoid bioaccessibility during the digestion of both individual and combined foods, and a positive correlation was found between the two sets of data. Furthermore, addition of cooked egg to the vegetable salad increased the in vitro bioaccessibility of lutein and lycopene, while the co-digestion of pan-fried salmon promoted the micellarization of 'alpha'-carotene, 'beta'-carotene and lutein, regardless of the digestion method employed. Three scientific papers were published in international peer-reviewed indexed journals with the results obtained during this period (AU)